1. Homologous Recombination in Real Time: DNA Strand Exchange by RecA
Thijn van der Heijden, Mauro Modesti, Susanne Hage, Roland Kanaar, Claire Wyman, Cees Dekker 
Molecular Cell 
Volume 30, Issue 4, Pages (May 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 RecA-Mediated DNA Strand Invasion and Exchange
(A) Schematic drawing of the magnetic tweezers setup. A DNA molecule is attached at one end to the bottom of the flow cell and at the other end to a magnetic bead. This molecule can be stretched and twisted by using a pair of magnets placed above the flow cell. The bead position, and thus the end-to-end distance of the DNA molecule, is determined by using video microscopy and image analysis. In this setup, the interaction of a RecA-coated single-stranded DNA filament with a homologous duplex DNA molecule can be followed in real time because binding induces a change in end-to-end distance of the tethered molecule.
(B) Rotation of the external magnets induces a change in end-to-end distance due to the formation of plectonemic supercoils (Strick et al., 1998). Thirty-five negative plectonemic supercoils were introduced at a stretching force of 0.5 pN before initiation of the strand invasion and exchange reaction. Upon binding, a RecA-coated single-stranded DNA nucleoprotein filament partially unwinds the target double-stranded DNA molecule and therefore amplifies the signal ΔL due to the removal of negative plectonemic supercoils and therefore shifting the center of the rotation dependency of the tethered molecule toward lower numbers.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Interaction of a RecA-Coated Single-Stranded DNA Nucleoprotein Filament with a Homologous Duplex DNA Molecule
(A) In the presence of ATPγS, the end-to-end distance of the double-stranded DNA tether increased monotonically before reaching a plateau at which no further elongation or shortening was observed (red). In the presence of ATP, however, the end-to-end distance displayed a smaller increase and finally returned to its original level (green).
(B) Control experiments in the absence of negative supercoiling (top curve) or lacking homology between a RecA-coated single-stranded DNA nucleoprotein filament and the double-stranded DNA molecule (bottom curve) did not result in changes of the end-to-end distance in the presence of ATPγS.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
The Rate of Strand Invasion and Joint-Molecule Formation for Different Nucleotide Cofactors, the Presence of Single-Stranded Binding Protein, and Partial Homology
The duration time of the length increase (τi) induced in the double-stranded DNA tether by its interaction with a RecA-coated single-stranded DNA nucleoprotein filament scales linearly with the length of homology for both ATP (i = 2) and ATPγS (i = 1), yielding a rate of strand invasion and joint-molecule formation of 2.25 ± 0.04 (green) and 2.11 ± 0.05 (red) nt/s, respectively. RecA-coated single-stranded DNA nucleoprotein filaments formed in the presence of SSB protein (triangle) show an interaction time similar to those assembled in the absence of SSB protein. Green and red represent measurements done in the presence of, respectively, ATPγS and ATP (data are represented as mean ± SEM, n = 5). The interaction time of RecA-coated single-stranded DNA with a 5′ heterologous tail corresponds to the length of homology between invading and target DNA (cross).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Change in Writhe during Joint-Molecule Formation
(A) After the formation of a joint molecule in the presence of ATPγS, a ΔWr was observed for the tethered duplex DNA molecule. In the case of 1055 nt of homology, the rotational offset was changed by −32 (red). After the formation of a joint molecule in the presence of ATP, however, the writhe was unchanged (green), independent of the length of homology used (see inset).
(B) The ΔWr observed with ATPγS scales linearly with the length of homology yielding a slope of ± turns per nucleotide, corresponding to the formation of a structure with 14.6 ± 0.2 bp per turn (red line). The data are represented as mean ± SEM (n = 5). The dotted line depicts the expected result for a complete unwinding during joint-molecule formation. The dashed line corresponds to the structure of a RecA-coated double-stranded DNA filament. The black solid line denotes the result of a RecA-bound three-strand structure derived from molecular modeling.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Schematic Representation of the Strand Invasion and Exchange Reaction by RecA
Interactions occur between a RecA-coated single-stranded DNA nucleoprotein filament (purple circles—black line) and a double-stranded DNA molecule (blue-red). During strand exchange, a window of synapsis of 79 ± 6 nt in length travels along the length of homology (toward the left in the cartoon, 5′ to 3′ with respect to the invading single strand). The direction of strand exchange and of RecA dissociation is consistent with established behavior of RecA and not determined specifically from our data. The final structure after strand exchange is obtained when RecA dissociates and the displaced single strand (blue) wraps around the newly formed duplex DNA. The exact structure of the D wrap has not yet been determined. Although drawn in a regular helix around the double strand, the displaced single strand is flexible and may not have a defined structure.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6 Change in Structure after Strand Exchange
(A) Restriction enzyme cleavage of the invading strand after D loop formation by RecA. (Left panel) D loop reactions were performed using a 5′ end-radiolabeled oligonucleotide (90-mer) homologous to a region of pUC19 plasmid encompassing an ApaLI restriction site. Reaction products were resolved by electrophoresis through a native agarose gel. (Right panel) After the D loop reaction, samples numbered as in the right panel before and after treatment with ApaLI were resolved by electrophoresis in a denaturing acrylamide gel.
(B) Addition of a restriction enzyme to the double-stranded DNA construct in the magnetic tweezers. Before strand exchange, addition of a restriction enzyme with its recognition sequence inside the region of homology led to the loss of bead tracking because the tethered DNA molecule was cut.
(C) After strand exchange by RecA in the presence of ATP, however, the restriction enzyme EcoRI did not cut the DNA tether but rather released the torsional constraint. The end-to-end distance was seen to increase due to the release of the negative plectonemic supercoils by rotation of one strand around the other. The inset shows the rotation-dependent behavior before (black) and after (red) the treatment with the restriction enzyme. Before introduction of the restriction enzyme in the flow cell, the tethered DNA molecule shows a torsionally constrained behavior, whereas after treatment the molecule is torsionally unconstrained, yielding the same end-to-end distance for various applied rotations.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions